Sign up to receive free email alerts when patent applications with chosen keywords are publishedSIGN UP

Abstract:

The present invention provides an isolated HIV-1 mutant and isolated
nucleic acid molecules comprising HIV-RT coding sequences harboring a
novel mutation in the S68 codon, and in particular, deletions of the S68
codon. This novel deletion reduces the sensitivity of HIV to various
nucleoside reverse transcriptase inhibitors. Methods of using this
mutation for selecting effective antiretroviral agents in vitro and in
vivo, methods for monitoring infection progression in HIV-infected
individuals and methods for avoiding the emergence of and/or to treat
individuals infected with HIV comprising mutations, including deletions,
at the S68 codon of HIV-RT are provided.

12. The method of claim 11, wherein if the HIV-infected individual was
undergoing an antiretroviral treatment prior to step (i), the treatment
is altered based on the determination step (ii).

13. (canceled)

14. The method of claim 12, wherein the alteration of treatment comprises
administering at least one antiretroviral agent that reduces or
eliminates RNA production by the HIV variant having a codon 68 deletion.

16. The method of claim 14, wherein the at least one antiretroviral agent
is selected from the group consisting of a protease inhibitor, a
non-nucleoside reverse transcriptase inhibitor, an HIV fusion inhibitor,
an HIV integrase inhibitor, an RNAse H inhibitor, a CD4 binding
inhibitor, a CXCR4 binding inhibitor and a CCR5 binding inhibitor.

17. The method of claim 14, wherein the at least one antiretroviral agent
is selected from the group consisting of AZT, DDI, DFDOC, D4T, DOT and
DDC.

18. The method of claim 11, wherein the absence of or decreasing
concentrations of detectable HIV sequences correlates positively with the
assessment that the antiretroviral agent is therapeutically effective in
treating the codon 68 mutant HIV.

19-42. (canceled)

Description:

[0001] This application is a divisional of U.S. patent application Ser.
No. 12/595,358, which is a national stage entry of PCT/US2008/004666,
filed Apr. 10, 2008, which claims priority from U.S. Provisional Patent
Application No. 60/922,838, filed Apr. 10, 2007, the contents of which
are hereby incorporated by reference in their entireties.

BACKGROUND OF THE INVENTION

[0003] In 1983, the etiological cause of AIDS was determined to be the
human immunodeficiency virus (HIV). In 1985, it was reported that the
synthetic nucleoside 3'-azido-3'-deoxythymidine (Zidovudine, AZT, ZDV)
inhibits the replication of human immunodeficiency virus by inhibiting in
its 5'-triphosphate form the HIV-1 reverse transcriptase (HIV-RT). HIV-RT
is active early in the viral replication cycle and is necessary for
continued viral replication. Currently, a total eight synthetic
nucleosides have been approved by the US FDA. These are: AZT (mentioned
above), 2',3'-dideoxyinosine (Videx, DDI), 2',3'-dideoxycytidine (DDC),
2',3'-dideoxy-2',3'-didehydrothymidine (stavudine, D4T),
cis-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane
(emtricitabine, FTC),
(-)-cis-2-hydroxymethyl-5-(cytosin-1-yl)-1,3-oxathiolane (Lamivudine,
3TC), (1S,4R)-4-[2-amino-6-(cyclopropyl-amino)-9H-purin-9-yl]-2-cyclopent-
ene-1-methanol succinate (abacavir, ABC), and the acyclic nucleotide
9-[(R)-2-[[bisaisopropoxycarbonyl)oxy]methoxy]phosphinyl]methoxy]propyl]a-
denine fumarate (tenofovir-DF, TDF). All nucleoside reverse transcriptase
inhibitors (NRTI) require phosphorylation to their triphosphate (TP)
forms, while metabolic activation of tenofovir requires phosphorylation
to its 12 diphosphate (tenofovir-DP). Such so-called NRTI mimic natural
nucleosides in the cell. After cellular phosphorylation to the
5'-triphosphate by cellular kinases, these synthetic nucleosides can be
incorporated into a growing strand of viral DNA, causing chain
termination due to the absence of the 3'-hydroxyl group found in natural
nucleosides that are used in the DNA chain elongation reaction catalyzed
by HIV-RT. NRTI therapies in HIV treatment are reviewed in Schinazi et
al., Antiviral Research 71:322-334 (2006)).

[0004] HIV shows high genetic variability in part as a result of its fast
viral replication cycle coupled with the high mutation rate of and active
recombinogenic characteristics of HIV-RT, especially during viral
replication in single cells co-infected by multiple different strains of
HIV. Drug-resistant variants of HIV can emerge after treatment with an
antiviral agent. Drug resistance most typically occurs by mutation of a
gene that encodes for an enzyme used in viral replication, and most
typically in the case of HIV, reverse transcriptase, protease, or DNA
polymerase. NRTI treatment of HIV-1 infected individuals often leads to
the emergence of mutations in the reverse transcriptase (RT). Less
frequently seen are codon insertions or deletions, either which add or
subtract three nucleotides and leave other codons in the correct coding
frame. Codon insertions (ins) and deletions (del) have been associated
with multi-drug resistance (MDR) in clinical samples obtained from HIV-1
infected individuals treated with antiretroviral agents (67del, 69del,
69ins, 70del).

[0005] The β3-β4 hairpin loop of the finger domain of RT is
thought to be directly involved in the interaction of the enzyme with its
substrates (the template-primer complex and the dNTP) (Tamalet et al.,
Virol. 270:310-316 (2000)). Genetic rearrangements in the β3-β4
loop have been found in patients extensively treated with anti-HIV drugs
and experiencing therapeutic failure (Tamalet, supra; Winters et al., J.
Virol. 74(22):10707-10713 (2000)).

[0006] The efficacy of a drug against HIV infection can be prolonged,
augmented, or restored by administering the compound in combination or
alternation with a second, and in particular a third, antiviral compound
that induces a different mutation from that caused by the principle drug.
Alternatively, the pharmacokinetics, biodistribution, or other parameter
of the drug can be altered by such combination or alternation therapy,
although this is not recommended for HIV infections. In general,
combination therapy is typically preferred over alternation therapy
because it induces multiple simultaneous pressures on the virus. One
cannot predict, however, what mutations will be induced in the HIV-1
genome by a given new drug, whether the mutation is permanent or
transient, or how an infected cell with a mutated HIV-1 sequence will
respond to therapy with other agents in combination or alternation. This
is exacerbated by the fact that there is a paucity of data on the
kinetics of drug resistance in long-term cell cultures treated with
modern antiretroviral agents.

[0007] HIV-1 variants resistant to AZT, DDI, 3TC, D4T, DDC, ABC or TDF
have been isolated from patients receiving long term monotherapy with
these drugs (Larder et al., Science 1989; 243:1731-4; St. Clair et al.,
Science 1991; 253:1557-9; and Fitzgibbon et al., Antimicrob. Agents
Chemother. 1992; 36:153-7; Schinazi, et al., Intl. Antiviral News 2000;
8:65-92). Mounting clinical evidence indicates that AZT and 3TC
resistance is a predictor of poor clinical outcome in both children and
adults. The rapid development of HIV-1 resistance to non-nucleoside
reverse transcriptase inhibitors (NNRTI) has also been reported both in
cell culture and in human clinical trials (Nunberg et al. J. Virol. 1991;
65(9):4887-92; Richman et al., Proc Natl Acad Sci (USA) 1991; 88:11241-5;
Mellors et al., Mol. Pharm. 1992; 41:446-51; Richman D D and the ACTG
164/168 Study Team. Second International HIV-1 Drug Resistance Workshop.
(Noordwijk, the Netherlands. 1993); and Saag et al., N Engl J Med 1993;
329:1065-1072). In the case of the NNRTI L'697,661, drug-resistant HIV-1
emerged within 2-6 weeks of initiating therapy in association with the
return of viremia to pretreatment levels. (Saag et al., supra).
Breakthrough viremia associated with the appearance of drug-resistant
strains has also been noted with other classes of HIV-1 inhibitors,
including protease, fusion and integrase inhibitors. This experience has
led to the realization that the potential for HIV-1 drug resistance must
be assessed early on in the preclinical evaluation of all new therapies
for HIV-1.

[0008] The emergence of resistant HIV strains during viral therapy has
presented a major challenge to delay, prevent or attenuate the onset of
resistance. Common resistance mutations, including thymidine associated
mutations (TAM), K65R and M184V are problematic in HIV drug development.
Mutations observed to emerge following exposure to various NRTI are
summarized in Schinazi et al., supra, 2006 (see Table 1). Novel NRTI are
under pre-clinical development that are good substrates for cellular
kinases, have high bioavailability (especially oral), reduced toxicity
and significant levels of activity against the commonly found
NRTI-resistant HIV-1 mutants, such as D67N, K70R, T215Y, K219Q, K65R and
M184V (Chu et al., J. Med. Chem. 48:3949-3952 (2005)).

[0009] 2',3'-Dideoxy-2',3'-didehydro-5-fluoro-cytidine (D4FC, DFC;
dexelvucitabine) is a known NRTI compound (see, e.g., EP 0 409 227 A2,
U.S. Pat. Nos. 5,703,058 and 5,905,070). Treatment with β-L-D4FC
rapidly selects for a mutation at codon 184 (methionine to valine) of the
reverse transcriptase region of the virus, resulting in a high level of
resistance to 3TC and FTC. β-D-D4FC, in contrast, is not
significantly cross-resistant to AZT, DDC, DDI, D4T, 3TC, (-)-FTC or
β-L-D4FC. β-D-D4FC treatment selects for HIV-1 variants having
mutations at codons 163L, K65R, K70N, K70E, or R172K of the HIV-RT region
of the virus (see also Hammond et al., Antimicrob. Agents Chemother.
49(9):3930-3932 (2005)). Thus, β-D-D4FC can be used generally as
salvage therapy for any HIV-infected individual that exhibits resistance
to other anti-HIV agents whose drug resistance patterns correlate with
mutations at codons different from those selected by 3-D-D4FC treatment.
Based on this, methods for treating HIV have been reported that involve
administering β-D-D4FC or its pharmaceutically acceptable salt or
prodrug in combination or alternation with a drug that selects for
variants having one or more mutations in HIV-1 at a location other than
codons 163L, K65R, K70N, K70E, or R172K (U.S. Pat. No. 7,115,584, and
Hammond et al.).

[0010] Current treatments for HIV infection are most often those referred
to as "highly active antiretroviral therapy" or HAART and involve
administering combinations ("cocktails") comprising at least three
drugs--two NRTI in combination with either a protease inhibitor or a
NNRTI. Results of studies on the emergence of drug resistance and
correlations between antiviral drugs and mutation patterns present in
selected HIV variant genes are useful in directing resistance testing of
viruses from HIV-infected individuals treated with antiviral agents such
as NRTI and in choosing combinations of nucleoside analogs for treatment
and prevention of drug resistant HIV. Characterization of these mutations
is key in determining potential cross-resistance and in HIV treatment
management. It is thus desirable to understand more about NRTI resistance
patterns and how they correlate with HIV genotypes and mutations in
essential HIV genes, such as HIV-RT.

SUMMARY OF THE INVENTION

[0011] The present invention addresses the problems above by identifying a
novel deletion in HIV-1 RT of the S68 codon ("S68del"; which may
alternatively be a deletion of the AGT codon 68 trinucleotide, or of the
adjacent +1 frameshift trinucleotide GTA) revealed during the selection
of virus with dexelvucitabine (DFC) in primary human lymphocytes. The
novel S68 deletion and the distinct multi-drug resistant phenotype it
imparts on HIV may be an important variable in NRTI multidrug resistance,
management of HIV-infected persons and improved treatment strategies.

[0012] The S68 deletion was investigated phenotypically against selected
antiviral agents for resistance and demonstrated resistance to several
clinically important NRTI. The S68del produced greater than 30-fold
increased resistance to DFC, lamivudine, emtricitabine, tenofovir,
abacavir and amdoxovir. As expected, the S68del demonstrated no
resistance to NNRTI and protease inhibitors.

[0013] Codon 68 mutants, and S68del in particular, are expected to precede
immunologic decline of an infected individual over time. Once the codon
68 mutation has been detected in plasma HIV RNA or lymphocytes of an
HIV-infected individual, a specific therapeutic regimen is considered. In
cases in which the HIV-infected individual is already undergoing
antiretroviral therapy, an alteration in the therapeutic regimen is
preferably considered.

[0014] In certain embodiments, the invention thus provides a nucleic acid
molecule comprising sequences encoding part or all of HIV-1 RT, the
HIV-RT sequences comprising a codon 68 mutation, provided that when the
codon 68 mutation is an S68 substitution, it occurs alone or in
combination with a mutation other than a K65R mutation. The invention
also provides a nucleic acid molecule comprising sequences encoding part
or all of HIV-1 RT, the HIV-RT sequences comprising a codon 68 mutation,
wherein the codon 68 mutation is the only mutation in the HIV-RT
sequences. The invention further provides a nucleic acid molecule
comprising sequences encoding part or all of HIV-1 RT, the HIV-RT
sequences comprising a codon 68 deletion wherein the S68 deletion removes
the codon 68 AGT trinucleotide, or wherein the S68 deletion removes the
GTA trinucleotide spanning codons 68 and 69. Preferably, isolated nucleic
acid molecules of the invention comprise a minimum of nine, preferably of
10-25 or more nucleotides so that they may be used as selective primers,
e.g., in nucleic acid amplification methods, or as probes in nucleic acid
hybridization techniques.

[0015] The invention also provides an isolated HIV-1 or HIV-2 comprising
any of the previously described nucleic acids.

[0016] The present invention also provides a method of evaluating the
effectiveness of an antiretroviral agent or preventing or treating HIV
infection of cells, comprising: (i) treating cells with an antiretroviral
agent; (ii) infecting cells with an HIV-1 (or HIV-2) having a codon 68
mutation in the reverse transcriptase coding sequence, provided that when
the codon 68 mutation is an S68 substitution, it is not in combination
with a K65R mutation; and (iii) determining the effect of the agent on
viral replication; wherein steps (i) and (ii) can be performed in any
order.

[0017] In one embodiment, the invention provides a method of selecting an
effective antiretroviral therapy for an HIV-infected person, the method
comprising: (i) collecting a plasma sample from an HIV-infected person
who is being treated with an antiretroviral agent; and (ii) determining
whether the plasma sample comprises nucleic acid encoding HIV-RT
sequences comprising a codon 68 mutation, provided that when the codon 68
mutation is an S68 substitution, it is not in combination with a K65R
mutation. In certain embodiments, the codon 68 mutation is determined by
a method comprising polymerase chain reaction. In certain of such
embodiments, the polymerase chain reaction is nested. In other such
embodiments, the polymerase chain reaction is real-time. In further
embodiments, the method comprising polymerase chain reaction utilizes
primers SK38: ATA ATC CAC CTA TCC CAG TAG GAG AAA T (SEQ ID NO: 1) and
SK39: TTT GGT CCT TGT CTT ATG TCC AGA ATG C (SEQ ID NO: 2).

[0018] It may be desirable after detecting the codon 68, e.g., S68del,
mutation to alter the course of the person's current treatment regimen to
include one or more antiretroviral agents that are effective in
inhibiting the replication of an HIV mutant comprising an S68 mutation,
e.g. S68del.

[0019] In another embodiment, the invention provides a method of selecting
an effective antiretroviral therapy for an HIV-infected individual,
comprising: (i) collecting lymphocytic cells from an HIV-infected
individual; and (ii) determining whether the cells comprise nucleic acid
encoding HIV-RT sequences comprising a codon 68 mutation, wherein if
HIV-RT sequences comprising a codon 68 mutation are present, an
antiretroviral therapy is selected which inhibits production of HIV-RT
codon 68 mutant variant RNA in the cells, provided that when the codon 68
mutation is an S68 substitution, it is not in combination with a K65R
mutation.

[0020] In certain embodiments, the invention provides a method of
selecting an effective antiretroviral therapy for an HIV-infected
individual, comprising: (i) collecting lymphocytic cells from an
HIV-infected individual; and (ii) determining whether the cells comprise
nucleic acid encoding HIV-RT sequences comprising a codon 68 mutation,
wherein if HIV-RT sequences comprising a codon 68 mutation are present,
an antiretroviral therapy is selected which inhibits production of HIV-RT
codon 68 mutant variant RNA in the cells, provided that when the codon 68
mutation is an S68 substitution, it is not in combination with a K65R
mutation, wherein if the HIV-infected individual was undergoing an
antiretroviral treatment prior to step (i), the treatment is altered
based on the determination step (ii).

[0021] In further embodiments, the invention provides a method for
evaluating the effectiveness to an HIV-infected individual of a selected
antiretroviral agent or therapy, the method comprising: (i) collecting a
sample from an HIV-infected individual; and (ii) determining whether the
sample comprises nucleic acid encoding HIV reverse transcriptase having a
mutation at codon 68, e.g., S68del, in which the presence of the mutation
correlates positively with refractoriness of the individual to the
selected antiretroviral therapy and, if the therapy remains unchanged, to
accelerated immunologic decline of the HIV-infected individual compared
to HIV-infected individuals who do not have the mutation.

[0022] In any of the above methods, the alteration of treatment may
comprise administering at least one antiretroviral agent that reduces or
eliminates RNA production by the HIV variant having a codon 68 mutation.
In certain embodiments, the at least one antiretroviral agent is selected
from a protease inhibitor, a non-nucleoside reverse transcriptase
inhibitor, an HIV fusion inhibitor, an HIV integrase inhibitor, an RNAse
H inhibitor, a CD4 binding inhibitor, a CXCR4 binding inhibitor and a
CCR5 binding inhibitor. In certain embodiments, the at least one
antiretroviral agent is selected from: AZT, DDI, DFDOC, D4T, DOT and DDC.

[0023] In any of the above methods, the absence of, or decreasing
concentrations of, detectable HIV sequence correlates positively with the
assessment that the antiretroviral agent is therapeutically effective in
treating a codon 68, e.g., S68del, mutation. Moreover, in this method,
the presence of, or increasing concentrations of, detectable HIV sequence
correlates positively with the assessment that the antiretroviral agent
is therapeutically ineffective and that a resistant virus has developed.

[0024] In another embodiment, the invention provides methods for
evaluating the effectiveness to an HIV-infected individual of treatment
with an antiretroviral agent or therapy prone to emergence of a codon 68
mutation, the method comprising (i) collecting a sample from an
HIV-infected individual before treatment with a selected antiretroviral
agent prone to emergence of a codon 68 mutation; (ii) collecting a sample
from the HIV-infected individual after treatment with the selected
antiretroviral agent; (iii) amplifying separately HIV-encoding nucleic
acid in the samples from (i) and (ii) with HIV primers; (iv) comparing
the HIV nucleic acid copy number in samples (i) and (ii), wherein a ratio
of HIV nucleic acid copy number in samples (i) and (ii) of greater than
about 2.5 to 1, 4 to 1, 5 to 1, 10 to 1 or more, correlates positively
with the assessment that the selected antiretroviral agent has not
selected an HIV-RT codon 68 mutation, e.g., S68del, and remains
therapeutically effective. In certain embodiments, such methods may
additionally or optionally (e.g., in step (iii)) comprise the use of HIV
primers that can distinguish between the presence and absence of a codon
68 mutation, e.g., S68del in HIV-RT.

[0025] In certain other embodiments, the invention provides methods for
evaluating the effectiveness to an HIV-infected individual of treatment
with an antiretroviral agent or therapy prone to emergence of a codon 68
mutation, the method comprising: (i) collecting at least one sample from
an HIV-infected individual at separate time intervals; (ii) amplifying
HIV-encoding nucleic acid in the separate samples using HIV primers;
(iii) measuring HIV nucleic acid copy numbers using the amplification
products of step (ii); and (iv) comparing the HIV nucleic acid copy
numbers in the samples collected during the course of the selected
treatment; whereby a statistically significant decline in HIV nucleic
acid copy numbers detected over the course of the treatment correlates
positively with the assessment that the selected antiretroviral agent has
not selected an HIV-RT codon 68 mutation, e.g., S68del, and remains
therapeutically effective. In certain embodiments, such methods may
additionally or optionally (e.g., in step (ii)) comprise the use of HIV
primers that can distinguish between the presence and absence of a codon
68 mutation, e.g., S68del in HIV-RT.

[0026] In any of the above products or methods of the invention, the
HIV-RT codon 68 mutation may be an S68 deletion that removes AGT or that
removes the GTA trinucleotide spanning codons 68 and 69.

[0027] In certain embodiments of the present invention, the HIV specific
primers used in the methods of the invention can distinguish between the
presence and absence of a HIV reverse transcriptase codon 68 mutation,
and more particularly, of the S68del mutation. Examples of such primers
include, without limitation, SK38 Primer: ATA ATC CAC CTA TCC CAG TAG GAG
AAA T (SEQ ID NO: 1) and SK39 Primer: TTT GGT CCT TGT CTT ATG TCC AGA ATG
C (SEQ ID NO: 2). The presence of amplified product may also be detected
with the SK19 probe: ATC CTG GGA TTA AAT AAA ATA GTA AGA ATG TAT AG (SEQ
ID NO: 3). Other HIV specific primers may easily be designed by those of
skill in the art that can detect and differentiate codon 68 mutations,
including those that distinguish between S68 substitutions and the codon
68 deletion referred to herein as "S68del".

[0028] The present invention also provides methods for treating a person
infected with HIV comprising the step of administering over time an
antiviral agent that does not select for an HIVvariant having a codon 68
mutation in the HIV-RT coding sequence. The codon 68 mutation is an S68
deletion that removes AGT or that removes the GTA trinucleotide spanning
codons 68 and 69. In certain embodiments, the antiretroviral agent is one
that is effective at inhibiting viral replication of an HIV-1 mutant
comprising an S68 mutation, e.g., an S68 deletion so that viral RNA
production is reduced or eliminated. In further embodiments, the
antiretroviral agent is selected from an HIV protease inhibitor such as
Lopinavir®; an HIV fusion inhibitor such as a CD4 binding inhibitor,
a CXCR4 binding inhibitor or a CCR5 binding inhibitor such as Maraviroc;
an HIV integrase inhibitor such as Raltegravir; an RNAseH inhibitor; an
NNRTI such as Sustiva®. In certain embodiments, the antiretroviral
agent is an NRTI that inhibits replication of an HIV-1 S68 mutant at
concentrations that are no more than 5-fold (and preferably no more than
2.5-fold) higher than the concentration of the agent required to inhibit
viral replication of wild-type HIV-1. In certain preferred embodiments,
the antiretroviral agent is selected from: AZT, DDI, DFDOC, D4T, DOT or
DDC. In yet other preferred embodiments, the antiretroviral agent is AZT.
In yet other preferred embodiments, the antiretroviral agent is DDI.

[0029] In other embodiments, the invention provides a kit comprising at
least one pair of primers designed to detect the presence of a codon 68
mutation in HIV-RT coding sequences. In further embodiments, the kit may
be designed to detect the codon 68 deletion that removes the AGT
trinucleotide encoding S68 or the GTA trinucleotide spanning codons 68
and 69. The kit may comprise at least one primer is selected from SK38
and SK39. The kit may further comprise a nucleic acid probe comprising or
consisting essentially of the following nucleic acid sequence (SK19): ATC
CTG GGA TTA AAT AAA ATA GTA AGA ATG TAT AG (SEQ ID NO: 3).

[0030] In certain embodiments, the invention provides a nucleic acid
product of priming with primers SK38 (SEQ ID NO: 1) and SK39 (SEQ ID NO:
2), wherein the nucleic acid product comprises sequences encoding HIV-1
RT, the HIV-RT sequences comprising a codon 68 mutation, provided that
when the codon 68 mutation is an S68 substitution, it is not in
combination with a K65R mutation.

[0032] In certain embodiments, the invention provides any of the above
nucleic acids or nucleic acid products attached to a solid support. In
further embodiments, the invention provides an array comprising any of
the above nucleic acids or nucleic acid products.

[0033] In certain embodiments, the invention provides use of an
antiretroviral agent to produce a composition useful in treating a
subject infected with HIV-1, wherein the antiretroviral agent is one
which does not select for an HIV-1 variant comprising a codon 68 mutation
in the HIV-RT coding sequence. In further embodiments, the codon 68
mutation is an S68 deletion that removes AGT or that removes the GTA
trinucleotide spanning codons 68 and 69. In certain embodiments, the
antiretroviral agent is selected from: a protease inhibitor, a
non-nucleoside reverse transcriptase inhibitor, an HIV fusion inhibitor,
an HIV integrase inhibitor, an RNAse H inhibitor, a CD4 binding
inhibitor, a CXCR4 binding inhibitor and a CCR5 binding inhibitor. In
further embodiments, the antiretroviral agent is selected from: AZT, DDI,
DFDOC, D4T, DOT and DDC.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034]FIG. 1 is a graph showing percent inhibition by DFC
(dexelvucitabine) and the amounts of DFC (in μm) used during isolation
of the S68del virus.

[0038] FIG. 5 is a graph showing the results of a heteropolymeric DNA
colorimetric RT assay performed with particle-derived S68del and M184V
RTs. FI50--fold increase in 50% effective concentration.

[0039]FIG. 6 is a graph showing the results of a heteropolymeric DNA
colorimetric RT assay performed with recombinant S68del, virally-derived
S68del and virally-derived M184V RTs. The recombinant S68del enzyme was
tested in two separate experiments in duplicate. The standard errors for
AZT-TP, (-) FTC-TP and DFC-TP were 0.1, 2 and 0.2, respectively.
TP--triphosphate.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0040] As used herein, a "phenotypic change" in an HIV mutant is one that
confers a statistically significant change in viral replication rates in
the presence of a select antiviral compound or agent, defined herein to
be at least a 2.5-fold, and preferably at least a 5-fold or higher
increase in EC50 compared to native virus in a constant cell line.
Similarly, a "resistant virus" refers to a virus that exhibits a
2.5-fold, and more typically, five- or greater fold increase in EC50
compared to naive virus in a constant cell line, including, but not
limited to PBM, MT2 or MT4 cells. The term "resistance" is used in its
most general sense and includes total resistance or partial resistance or
decreased sensitivity to a nucleoside analogue.

[0041] The term "D-D4FC" is used herein interchangeably with the terms
β-D-D4FC and DFC, below. The S68del mutation was selected in and
confers resistance to DFC, and is expected to confer resistance to
certain DFC prodrugs.

[0042] As used herein, the term "prodrug" refers to the 5' and N4
acylated, alkylated, or phosphorylated (including mono, di, and
triphosphate esters as well as stabilized phosphates and phospholipid)
derivatives of D-D4FC. In one embodiment, the acyl group is a carboxylic
acid ester in which the non-carbonyl moiety of the ester group is
selected from straight, branched, or cyclic alkyl, alkoxyalkyl including
methoxymethyl, aralkyl including benzyl, aryloxyalkyl including
phenoxymethyl, aryl including phenyl optionally substituted by halogen,
alkyl, alkyl or alkoxy, sulfonate esters such as alkyl or aralkyl
sulphonyl including methanesulfonyl, trityl or monomethoxytrityl,
substituted benzyl, trialkylsilyl, or diphenylmethylsilyl. Aryl groups in
the esters optimally comprise a phenyl group. The alkyl group can be
straight, branched or cyclic and is preferably C1 to C18.

[0043] As used herein, "S68del" refers to a novel deletion of sequences at
codon 68 of HIV-RT, e.g., HIV-1 RT encoding sequences, which may
alternatively be a deletion of the AGT codon 68 trinucleotide, or of the
adjacent +1 frameshift trinucleotide GTA.

[0044] As used herein, "a codon 68 mutation" refers to a codon 68
substitution or S68del, but not a larger deletion that encompasses
S68del.

[0045] The following terms, unless otherwise indicated, shall be
understood to have the following meanings:

[0046] The term "allelic variant" refers to one of two or more alternative
naturally-occurring forms of a gene, wherein each gene possesses a unique
nucleotide sequence. In a preferred embodiment, different alleles of a
given gene have similar or identical biological properties.

[0047] The term "polynucleotide" or "nucleic acid molecule" refers to a
polymeric form of nucleotides of at least 10 bases in length. The term
includes DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNA
molecules (e.g., mRNA or synthetic RNA), as well as analogs of DNA or RNA
containing non-natural nucleotide analogs, non-native internucleoside
bonds, or both. The nucleic acid can be in any topological conformation.
For instance, the nucleic acid can be single-stranded, double-stranded,
triple-stranded, quadruplexed, partially double-stranded, branched,
hairpinned, circular, or in a padlocked conformation. The term includes
single and double stranded forms of DNA.

[0048] Unless otherwise indicated, a "nucleic acid comprising SEQ ID NO:
X" refers to a nucleic acid, at least a portion of which has either (i)
the sequence of SEQ ID NO: X, or (ii) a sequence complementary to SEQ ID
NO: X. The choice between the two is dictated by the context. For
instance, if the nucleic acid is used as a probe, the choice between the
two is dictated by the requirement that the probe be complementary to the
desired target.

[0049] An "isolated" or "substantially pure" nucleic acid or
polynucleotide (e.g., an RNA, DNA or a mixed polymer) is one which is
substantially separated from other cellular components that naturally
accompany the native polynucleotide in its natural host cell, e.g.,
ribosomes, polymerases, and genomic sequences with which it is naturally
associated. The term embraces a nucleic acid or polynucleotide that (1)
has been removed from its naturally occurring environment, provided that
it is not an unidentified member of a library which has not been
separated from other members, (2) is not associated with all or a portion
of a polynucleotide in which the "isolated polynucleotide" is found in
nature, (3) is operatively linked to a polynucleotide which it is not
linked to in nature, or (4) does not occur in nature. The term "isolated"
or "substantially pure" also can be used in reference to recombinant or
cloned DNA isolates, chemically synthesized polynucleotide analogs, or
polynucleotide analogs that are biologically synthesized by heterologous
systems.

[0050] However, "isolated" does not necessarily require that the nucleic
acid or polynucleotide so described has itself been physically removed
from its native environment. For instance, an endogenous nucleic acid
sequence in the genome of an organism is deemed "isolated" herein if a
heterologous sequence (i.e., a sequence that is not naturally adjacent to
this endogenous nucleic acid sequence) is placed adjacent to the
endogenous nucleic acid sequence, such that the expression of this
endogenous nucleic acid sequence is altered. By way of example, a
non-native promoter sequence can be substituted (e.g., by homologous
recombination) for the native promoter of a gene in the genome of a human
cell, such that this gene has an altered expression pattern. This gene
would now become "isolated" because it is separated from at least some of
the sequences that naturally flank it.

[0051] A nucleic acid is also considered "isolated" if it contains any
modifications that do not naturally occur to the corresponding nucleic
acid in a genome. For instance, an endogenous coding sequence is
considered "isolated" if it contains an insertion, deletion or a point
mutation introduced artificially, e.g., by human intervention. An
"isolated nucleic acid" also includes a nucleic acid integrated into a
host cell chromosome at a heterologous site, a nucleic acid construct
present as an episome. Moreover, an "isolated nucleic acid" can be
substantially free of other cellular material, or substantially free of
culture medium when produced by recombinant techniques, or substantially
free of chemical precursors or other chemicals when chemically
synthesized.

[0052] As used herein, the phrase "degenerate variant" of a reference
nucleic acid sequence encompasses nucleic acid sequences that can be
translated, according to the standard genetic code, to provide an amino
acid sequence identical to that translated from the reference nucleic
acid sequence.

[0053] The term "percent sequence identity" or "identical" in the context
of nucleic acid sequences refers to the residues in the two sequences
which are the same when aligned for maximum correspondence. The length of
sequence identity comparison may be over a stretch of at least about nine
nucleotides, usually at least about 20 nucleotides, more usually at least
about 24 nucleotides, typically at least about 28 nucleotides, more
typically at least about 32 nucleotides, and preferably at least about 36
or more nucleotides. There are a number of different algorithms known in
the art which can be used to measure nucleotide sequence identity. For
instance, polynucleotide sequences can be compared using FASTA, Gap or
Bestfit, which are programs in Wisconsin Package Version 10.0, Genetics
Computer Group (GCG), Madison, Wis. FASTA provides alignments and percent
sequence identity of the regions of the best overlap between the query
and search sequences (Pearson, 1990). For instance, percent sequence
identity between nucleic acid sequences can be determined using FASTA
with its default parameters (a word size of 6 and the NOPAM factor for
the scoring matrix) or using Gap with its default parameters as provided
in GCG Version 6.1, herein incorporated by reference.

[0054] The term "substantial homology" or "substantial similarity," when
referring to a nucleic acid or fragment thereof, indicates that, when
optimally aligned with appropriate nucleotide insertions or deletions
with another nucleic acid (or its complementary strand), there is
nucleotide sequence identity in at least about 50%, more preferably 60%
of the nucleotide bases, usually at least about 70%, more usually at
least about 80%, preferably at least about 90%, and more preferably at
least about 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% of the nucleotide
bases, as measured by any well-known algorithm of sequence identity, such
as FASTA, BLAST or Gap.

[0055] Alternatively, substantial homology or similarity exists when a
nucleic acid or fragment thereof hybridizes to another nucleic acid, to a
strand of another nucleic acid, or to the complementary strand thereof,
under stringent hybridization conditions. "Stringent hybridization
conditions" and "stringent wash conditions" in the context of nucleic
acid hybridization experiments depend upon a number of different physical
parameters. Nucleic acid hybridization will be affected by such
conditions as salt concentration, temperature, solvents, the base
composition of the hybridizing species, length of the complementary
regions, and the number of nucleotide base mismatches between the
hybridizing nucleic acids, as will be readily appreciated by those
skilled in the art. One having ordinary skill in the art knows how to
vary these parameters to achieve a particular stringency of
hybridization.

[0056] In general, "stringent hybridization" is performed at about
25° C. below the thermal melting point (Tm) for the specific DNA
hybrid under a particular set of conditions. "Stringent washing" is
performed at temperatures about 5° C. lower than the Tm for the
specific DNA hybrid under a particular set of conditions. The Tm is the
temperature at which 50% of the target sequence hybridizes to a perfectly
matched probe. See Sambrook et al., supra, page 9.51, hereby incorporated
by reference. For purposes herein, "high stringency conditions" are
defined for solution phase hybridization as aqueous hybridization (i.e.,
free of formamide) in 6×SSC (where 20×SSC contains 3.0 M NaCl
and 0.3 M sodium citrate), 1% SDS at 65° C. for 8-12 hours,
followed by two washes in 0.2×SSC, 0.1% SDS at 65° C. for 20
minutes. It will be appreciated by the skilled worker that hybridization
at 65° C. will occur at different rates depending on a number of
factors including the length and percent identity of the sequences which
are hybridizing.

[0057] The nucleic acids (also referred to as polynucleotides) of this
invention may include both sense and antisense strands of RNA, cDNA,
genomic DNA, and synthetic forms and mixed polymers of the above. They
may be modified chemically or biochemically or may contain non-natural or
derivatized nucleotide bases, as will be readily appreciated by those of
skill in the art. Such modifications include, for example, labels,
methylation, substitution of one or more of the naturally occurring
nucleotides with an analog, internucleotide modifications such as
uncharged linkages (e.g., methyl phosphonates, phosphotriesters,
phosphoramidates, carbamates, etc.), charged linkages (e.g.,
phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g.,
polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators,
alkylators, and modified linkages (e.g., alpha anomeric nucleic acids,
etc.) Also included are synthetic molecules that mimic polynucleotides in
their ability to bind to a designated sequence via hydrogen bonding and
other chemical interactions. Such molecules are known in the art and
include, for example, those in which peptide linkages substitute for
phosphate linkages in the backbone of the molecule.

[0058] The term "mutated" when applied to nucleic acid sequences means
that nucleotides in a nucleic acid sequence may be inserted, deleted or
changed compared to a reference nucleic acid sequence. A single
alteration may be made at a locus (a point mutation) or multiple
nucleotides may be inserted, deleted or changed at a single locus. In
addition, one or more alterations may be made at any number of loci
within a nucleic acid sequence. A nucleic acid sequence may be mutated by
any method known in the art including but not limited to mutagenesis
techniques such as "error-prone PCR" (a process for performing PCR under
conditions where the copying fidelity of the DNA polymerase is low, such
that a high rate of point mutations is obtained along the entire length
of the PCR product. See, e.g., Leung, D. W., et al., Technique, 1, pp.
11-15 (1989) and Caldwell, R. C. & Joyce G. F., PCR Methods Applic., 2,
pp. 28-33 (1992)); and "oligonucleotide-directed mutagenesis" (a process
which enables the generation of site-specific mutations in any cloned DNA
segment of interest. See, e.g., Reidhaar-Olson, J. F. & Sauer, R. T., et
al., Science, 241, pp. 53-57 (1988)).

[0059] The term "vector" as used herein is intended to refer to a nucleic
acid molecule capable of transporting another nucleic acid to which it
has been linked. One type of vector is a "plasmid", which refers to a
circular double stranded DNA loop into which additional DNA segments may
be ligated. Other vectors include cosmids, bacterial artificial
chromosomes (BAC) and yeast artificial chromosomes (YAC). Another type of
vector is a viral vector, wherein additional DNA segments may be ligated
into the viral genome (discussed in more detail below). Certain vectors
are capable of autonomous replication in a host cell into which they are
introduced (e.g., vectors having an origin of replication which functions
in the host cell). Other vectors can be integrated into the genome of a
host cell upon introduction into the host cell, and are thereby
replicated along with the host genome. Moreover, certain preferred
vectors are capable of directing the expression of genes to which they
are operatively linked. Such vectors are referred to herein as
"recombinant expression vectors" (or simply, "expression vectors").

[0060] "Operatively linked" expression control sequences refers to a
linkage in which the expression control sequence is contiguous with the
gene of interest to control the gene of interest, as well as expression
control sequences that act in trans or at a distance to control the gene
of interest.

[0061] The term "expression control sequence" as used herein refers to
polynucleotide sequences which are necessary to affect the expression of
coding sequences to which they are operatively linked. Expression control
sequences are sequences which control the transcription,
post-transcriptional events and translation of nucleic acid sequences.
Expression control sequences include appropriate transcription
initiation, termination, promoter and enhancer sequences; efficient RNA
processing signals such as splicing and polyadenylation signals;
sequences that stabilize cytoplasmic mRNA; sequences that enhance
translation efficiency (e.g., ribosome binding sites); sequences that
enhance protein stability; and when desired, sequences that enhance
protein secretion. The nature of such control sequences differs depending
upon the host organism; in prokaryotes, such control sequences generally
include promoter, ribosomal binding site, and transcription termination
sequence. The term "control sequences" is intended to include, at a
minimum, all components whose presence is essential for expression, and
can also include additional components whose presence is advantageous,
for example, leader sequences and fusion partner sequences.

[0062] The term "recombinant host cell" (or simply "host cell"), as used
herein, is intended to refer to a cell into which a recombinant vector
has been introduced. It should be understood that such terms are intended
to refer not only to the particular subject cell but to the progeny of
such a cell. Because certain modifications may occur in succeeding
generations due to either mutation or environmental influences, such
progeny may not, in fact, be identical to the parent cell, but are still
included within the scope of the term "host cell" as used herein. A
recombinant host cell may be an isolated cell or cell line grown in
culture or may be a cell which resides in a living tissue or organism.

[0063] The term "peptide" as used herein refers to a short polypeptide,
e.g., one that is typically less than about 50 amino acids long and more
typically less than about 30 amino acids long. The term as used herein
encompasses analogs and mimetics that mimic structural and thus
biological function.

[0064] Unless otherwise defined, all technical and scientific terms used
herein have the same meaning as commonly understood by one of ordinary
skill in the art to which this invention pertains. Exemplary methods and
materials are described below, although methods and materials similar or
equivalent to those described herein can also be used in the practice of
the present invention and will be apparent to those of skill in the art.
All publications and other references mentioned herein are incorporated
by reference in their entirety. In case of conflict, the present
specification, including definitions, will control. The materials,
methods, and examples are illustrative only and not intended to be
limiting.

S68del HIV-1 Mutant

[0065] In the present invention, the inventors have identified a mutant
HIV-1 with a novel mutation in the HIV reverse transcriptase (RT) coding
sequences. The novel mutation is a deletion in HIV-1 RT of the S68 codon
("S68del"). The S68del mutation was revealed during the selection of
virus to dexelvucitabine (DFC) in primary human lymphocytes. The mutation
reduces the sensitivity of HIV to nucleoside analogues to varying
extents. The identification of this HIV-RT mutant and characterization of
its phenotypic properties (e.g., its resistance and sensitivity profiles)
are important for the development of assays to monitor NRTI treatment
regimens and to screen for agents which can overcome the effects of the
mutation.

[0066] HIV codon 68 mutants of the invention may be in isolated form such
that they have undergone at least one purification step away from
naturally occurring body fluids. Alternatively, the mutants may be
maintained in isolated body fluid. In certain embodiments, the mutants
may be in RNA or DNA form. The present invention also includes infectious
molecular clones and nucleic acids comprising the genome or parts thereof
from an HIV harboring a codon 68 mutation, such as, e.g., S68del.

[0068] The invention provides a nucleic acid molecule comprising sequences
encoding HIV-1 RT, and in some embodiments, encoding fragments of HIV-1
RT, the HIV-RT sequences comprising a codon 68 mutation. In certain
embodiments, when the codon 68 mutation is an S68 substitution, it occurs
alone or in combination with a mutation other than a K65R mutation. The
invention also provides a nucleic acid molecule comprising sequences
encoding part or all of HIV-1 RT, the HIV-RT sequences comprising a codon
68 mutation, wherein the codon 68 mutation is the only mutation in the
HIV-RT sequences. The invention further provides a nucleic acid molecule
comprising sequences encoding HIV-1 RT, the HIV-RT sequences comprising a
codon 68 deletion wherein the S68 deletion removes the codon 68 AGT
trinucleotide, or wherein the S68 deletion removes the GTA trinucleotide
spanning codons 68 and 69. Preferably, isolated nucleic acid molecules of
the invention comprise a minimum of nine, preferably of 10-25 or more
nucleotides so that they may be used as selective primers, e.g., in
nucleic acid amplification methods, or as probes in nucleic acid
hybridization techniques.

[0069] The invention also provides vectors comprising any of the
previously described nucleic acids. Such vectors may be RNA or DNA based,
and may be replicative or integrative vectors, expression vectors
(transient or stable), viral vectors and the like.

[0070] The invention also provides an isolated HIV, such as HIV-1 or
HIV-2, comprising any of the previously described nucleic acids.

[0071] The invention also provides a host cell comprising any one of the
nucleic acids or isolated HIV of the invention.

[0072] The present invention also provides HIV specific primers which may
be used in conjunction with the methods and kits of the invention. In
certain embodiments, a primer of the invention can be used to amplify a
region of HIV-RT comprising codon 68 and preferably, can be used to
distinguish between the presence and absence of an HIV reverse
transcriptase codon 68 mutation, and more particularly, of the S68del
mutation. Examples of such primers include, without limitation, SK38
Primer: ATA ATC CAC CTA TCC CAG TAG GAG AAA T (SEQ ID NO: 1) and SK39
Primer: TTT GGT CCT TGT CTT ATG TCC AGA ATG C (SEQ ID NO: 2). The
presence of amplified product may also be detected with the SK19 probe:
ATC CTG GGA TTA AAT AAA ATA GTA AGA ATG TAT AG (SEQ ID NO: 3). The
invention thus also provides a nucleic acid probe that can be used to
distinguish between the presence and absence of an HIV reverse
transcriptase codon 68 mutation, and more particularly, of the S68del
mutation. Other HIV specific primers and probes may easily be designed by
those of skill in the art that can detect and differentiate codon 68
mutations, including those that distinguish between S68 substitutions and
the codon 68 deletion referred to herein as "S68del".

Methods of Evaluating Effectiveness of an Antiretroviral Agent and of
Monitoring the Progression of HIV-Infection

[0073] The present invention provides methods for monitoring the clinical
progression of human immunodeficiency virus (HIV) infection and its
response to selected antiviral therapies. It involves, in certain
embodiments, measuring HIV nucleic acid copy number in plasma or
lymphocytes (e.g., peripheral blood mononuclear cells) derived from an
HIV-infected person. Such measurements are performed, e.g., using RT (to
copy RNA to cDNA where HIV RNA is being assessed) and quantitative real
time polymerase chain reaction (PCR) assays, to assess an individual's
HIV viral load. Direct measurement of viral loads enables one to evaluate
the therapeutic effect of one or more select antiretroviral agents, and
therapies comprising administering such agents. Genotypic analyses of HIV
nucleic acid sequences and more specifically, of mutations that emerge
during treatment with select antiretroviral agents allow one to
understand how viral genotypic changes correlate with viral phenotypic
characteristics and reveal how emerging mutations affect viral RNA levels
in the presence of select antiretroviral agents. When a correlation
between viral genotype and phenotypic characteristics is demonstrated, it
provides useful methods that may be used, alone or in combination, for
predicting clinical outcome and thus for improving patient management and
care.

[0074] In working examples disclosed herein, a novel mutation of HIV RT
(deletion of AGT at codon S68, "S68del") was selected in a
dexelvucitabine (DFC)-treated primary human lymphocytes after 14 weeks of
culture. The S68del mutation became the dominant virus by week 19 based
on population sequence and clonal analysis. The S68del was investigated
phenotypically against selected antiviral agents and cross resistance
determined by drug susceptibility assays. The S68del correlated with
refractoriness not only to DFC treatment (during which it emerged), but
also to numerous other clinically important NSRI. Codon insertions and
deletions have been associated with multi-drug resistance (MDR) in
clinical samples from antiretroviral treated individuals (e.g., 76del,
69del, 69ins, 70del) but the S68del has never been reported.

[0075] Mutations at codon 68 of HIV-RT, and more particularly, of S68del,
correlate with resistance to certain antiretroviral therapies, including
DFC and lamivudine, emtricitabine, tenofovir, abacavir and amdoxovir
monotherapies. Codon 68 mutants, and S68 del in particular, are expected
to precede immunologic decline over time, e.g., by one or more, or 2-6 or
more months. Once mutation such as a deletion at codon 68 has been
detected in plasma HIV RNA or lymphocytes of an HIV-infected individual,
a specific therapeutic regimen is considered. In cases in which the
HIV-infected individual is already undergoing antiretroviral therapy, an
alteration in the therapeutic regimen is preferably considered. For
example, adding, subtracting or changing agents to overcome resistance to
the treatment may be considered.

[0076] In certain embodiments of the invention, quantitative real-time PCR
or other real-time sequence detection assays may be used to detect and
monitor the absolute concentrations and relative proportions of virus
with mutations at codon 68 (e.g., S68del) of HIV-RT, a mutation which
correlates with resistance to therapy with DFC and cross-resistance to a
number of other NRTI, including but not limited to lamivudine,
emtricitabine, tenofovir, abacavir and amdoxovir. When mutation at codon
68 (e.g., S68del) has been detected in a person undergoing monotherapy
with DFC or any other antiretroviral agent, an alteration in the
therapeutic regimen is preferably considered for effective patient
management. Such alteration may include, for example, combination
therapy, e.g. adding AZT to the DFC or other antiretroviral agent under
which the mutation is detected, or combination therapy with another
antiviral agent that is effective in inhibiting viral replication of an
HIV harboring the codon 68 mutation (e.g., S68del).

[0077] Accordingly, in one particular embodiment, the invention provides a
method for evaluating the effectiveness to an HIV-infected individual of
a selected antiretroviral agent or therapy, the method comprising: (i)
collecting a sample from an HIV-infected person treated with an
antiretroviral agent (the agent may be a antiretroviral compound or may
be a composition comprising at least one antiretroviral compound); (ii)
amplifying (e.g., by PCR) the HIV-encoding nucleic acid in the sample
using HIV specific primers; and (iii) testing for the presence of HIV
specific nucleic acid sequences in the amplification product of (ii). The
sample from the HIV-infected individual may be derived, e.g., from plasma
or lymphocytic cells, such as PMB cells, of the infected person. When the
sample is plasma, the HIV encoded nucleic acid is predominantly viral
RNA. When the sample is derived from lymphocytes, the HIV encoded nucleic
acid is predominantly proviral DNA. In certain preferred embodiments, the
HIV specific primers used in (ii) can distinguish between the presence
and absence of a HIV reverse transcriptase codon 68 mutation, and more
particularly, of the S68del mutation. In certain other separate
embodiments, step (iii), and not necessarily step (ii), distinguishes
between the presence and absence of a codon 68 mutation, e.g., S68del.

[0078] In a further embodiment, the invention provides a method for
evaluating the effectiveness to an HIV-infected individual of a selected
antiretroviral agent or therapy, the method comprising: (i) collecting a
sample from an HIV-infected individual; and (ii) determining whether the
sample comprises nucleic acid encoding HIV reverse transcriptase having a
mutation at codon 68, e.g., S68del, in which the presence of the mutation
correlates positively with refractoriness of the individual to the
selected antiretroviral therapy and, if the therapy remains unchanged, to
accelerated immunologic decline of the HIV-infected individual compared
to HIV-infected individuals who do not have the mutation. The sample from
the HIV-infected individual may be, e.g., plasma or lymphocytic cells
such as PBM cells. When the sample is plasma, the HIV encoded nucleic
acid is predominantly viral RNA. When the sample is lymphocytic cells,
the HIV encoded nucleic acid is predominantly proviral DNA.

[0079] In any of the above methods, the absence of, or decreasing
concentrations of, detectable HIV sequence correlates positively with the
assessment that the antiretroviral agent is therapeutically effective in
treating a codon 68, e.g., S68del, mutation. Moreover, in this method,
the presence of, or increasing concentrations of, detectable HIV sequence
correlates positively with the assessment that the antiretroviral agent
is therapeutically ineffective.

[0080] In another embodiment, the invention provides methods for
evaluating the effectiveness to an HIV-infected individual of treatment
with a selected antiretroviral agent, comprising (i) collecting a sample
from an HIV-infected individual before treatment with a selected
antiretroviral agent; (ii) collecting a sample from the HIV-infected
individual after treatment with the selected antiretroviral agent; (iii)
amplifying separately HIV-encoding nucleic acid in the samples from (i)
and (ii) with HIV primers; (iv) comparing the HIV nucleic acid copy
number in samples (i) and (ii), wherein a ratio of HIV nucleic acid copy
number in samples (i) and (ii) of greater than about 2.5 to 1, 4 to 1, 5
to 1, 10 to 1 or more, correlates positively with the assessment that the
selected antiretroviral agent is therapeutically effective. The sample
from the HIV-infected individual may be, e.g., plasma or lymphocytic
cells such as PBM cells. When the sample is plasma, the HIV encoded
nucleic acid is predominantly viral RNA. When the sample is lymphocytic
cells, the HIV encoded nucleic acid is predominantly proviral DNA. In
certain embodiments, such methods may additionally or optionally (e.g.,
in step (iii)) comprise the use of HIV primers that can distinguish
between the presence and absence of a codon 68 mutation, e.g., S68del in
HIV-RT.

[0081] Methods such as those described above may be modified to further
include one or more steps of collecting and analyzing a sample from an
HIV-infected individual so as to monitor the efficacy of the course of
the individual's treatment over time and to make changes to the person's
treatment regimen as needed, based on correlations derived from measuring
and comparing HIV genomic mutations, and specific nucleic acid levels
before and after treatment with a selected antiretroviral agent or
therapy.

[0082] Accordingly, in certain embodiments, the invention provides methods
for evaluating the effectiveness to an HIV-infected individual of
treatment with a selected antiretroviral agent treatment prone to
emergence of a codon 68 mutation, the method comprising: (i) collecting
at least one sample from an HIV-infected individual at separate time
intervals; (ii) amplifying HIV-encoding nucleic acid in the separate
samples using HIV primers; (iii) measuring HIV nucleic acid copy numbers
using the amplification products of step (ii); and (iv) comparing the HIV
nucleic acid copy numbers in the samples collected during the course of
the selected treatment; whereby a statistically significant decline in
HIV nucleic acid copy numbers detected over the course of the treatment
correlates positively with the assessment that the selected
antiretroviral agent has not selected for a HIV-RT codon 68 mutation,
e.g., S8del, and remains therapeutically effective. In certain
embodiments, such methods may additionally or optionally (e.g., in step
(iii)) comprise the use of HIV primers that can distinguish between the
presence and absence of a codon 68 mutation, e.g., S68del in HIV-RT. The
sample from the HIV-infected individual may be, e.g., plasma or
lymphocytic cells such as PBM cells. When the sample is plasma, the HIV
encoded nucleic acid is predominantly viral RNA. When the sample is
lymphocytic cells, the HIV encoded nucleic acid is predominantly proviral
DNA.

[0083] In other embodiments of the invention, the methods may be used to
detect mutations at codon 68 of HIV-RT, e.g., S68del, which correlate
with resistance to a selected antiretroviral therapy and which precede
immunologic decline. Accordingly, the present invention provides methods
for evaluating the effectiveness of a selected antiretroviral therapy to
an HIV-infected individual, the method comprising: (i) collecting a
sample from an HIV-infected individual who is being treated with an
antiretroviral agent; and (ii) determining (for example, using
quantitative, real time PCR) whether the sample comprises nucleic acid
encoding HIV-RT having a mutation at codon 68, e.g., S68del, in which the
presence of the mutation correlates positively with immunologic decline
of the individual within at least one, two, three four, five, six or ten
or more months. Under such circumstances, the HIV-infecting individual
has become, via the mutation, resistant to the selected antiretroviral
agent. It may be desirable after detecting the codon 68, e.g., S68del,
mutation to alter the course of the person's current treatment regimen.
The altered treatment regimen may be a complete exchange of
antiretroviral compounds or agents or may comprise adding one or more
additional antiretroviral agents to the HIV-infected individual's current
treatment regimen. For example, if the individual was being treated with
DFC when the mutation arose, the individual's therapeutic regimen may
desirably be altered, within about a six to twelve month period of the
mutation's occurrence, by either (i) changing to a different
antiretroviral agent, such as zidovudine (AZT) and stopping DFC
treatment; (ii) increasing the dosage of DFC (which is often less
desirable); or (iii) adding another antiretroviral agent, such as
zidovudine (AZT); to the person's therapeutic regimen; or any combination
thereof. The effectiveness of the modification in therapy may be
evaluated, as set forth above, by monitoring HIV nucleic acid copy
numbers after the treatment change. A subsequent decrease in circulating
HIV RNA copy number, for example, correlates positively with the
effectiveness of the new treatment regimen.

[0084] Because the mutation at the codon 68, e.g., S68del, may appear
first in plasma HIV RNA and only later in lymphocytic cell proviral DNA,
monitoring the time course of appearance of the codon 68 mutation in
proviral DNA may be desirable. Accordingly, the present invention also
provides methods for evaluating the effectiveness to an HIV-infected
person of antiretroviral therapy, the method comprising: (i) collecting
lymphocytic cells from an HIV-infected person who is being treated with
an antiretroviral agent; and (ii) determining whether the cells comprise
proviral HIV DNA comprising a mutation at codon 68 (e.g., S68del), in
which the presence of the mutation correlates positively with immunologic
decline of the individual over time. (The time depends in part on how
much sooner the mutation identified in the individual's plasma HIV RNA
precedes the mutation being detected in proviral DNA, which may be
anywhere from about 1 to about 5, 6, 7, 8, 9, 10 or more months).
Detection of the codon 68, e.g., S68del, mutation in proviral DNA is an
indicator of immunologic decline and alteration of the person's
therapeutic regimen is desirable.

[0085] In a specific embodiment of the invention, a method for evaluating
the effectiveness to an HIV-infected person of DFC therapy is provided,
the method comprising: (i) collecting a sample (e.g., plasma) from an
HIV-infected person who is being treated with DFC; (ii) amplifying the
HIV-encoding RNA in the sample by converting the RNA to cDNA and
amplifying HIV sequences using HIV primers and PCR, for example; and
(iii) testing for the presence of HIV sequence in the amplification
product of (ii), wherein the absence of detectable HIV sequence
correlates positively with the conclusion that DFC is therapeutically
effective and the presence of detectable HIV sequence correlates
positively with the conclusion that DFC is therapeutically ineffective.
In other embodiments, the sample from the HIV-infected individual is
derived from or comprises lymphocytic cells and step (ii) comprises
amplifying HIV proviral DNA sequences without a required conversion of
RNA to cDNA. In preferred embodiments of the above methods, the HIV
primers used comprise SK38 Primer: ATA ATC CAC CTA TCC CAG TAG GAG AAA T
(SEQ ID NO: 1) and SK39 Primer: TTT GGT CCT TGT CTT ATG TCC AGA ATG C
(SEQ ID NO: 2), and/or the presence of HIV sequence is detected using,
e.g., an enzyme-linked assay (e.g., a colorimetric or fluorescence based
assay). The presence of the amplified product may also be detected with
the SK19 probe: ATC CTG GGA TTA AAT AAA ATA GTA AGA ATG TAT AG (SEQ ID
NO: 3). Similar methods in which the HIV copy number is measured are also
provided.

[0086] Another specific embodiment of the invention provides a method for
evaluating the effectiveness to an HIV-infected individual of DFC
therapy, the method comprising: (i) collecting a sample (e.g., plasma)
from an HIV-infected individual who is being treated with DFC; (ii)
amplifying the HIV-encoding RNA in the sample by converting the RNA to
cDNA and amplifying HIV sequences using HIV primers and PCR to produce a
PCR amplification product that comprises a portion of the HIV-RT gene
containing codon 68 (e.g. SK38 Primer: ATA ATC CAC CTA TCC CAG TAG GAG
AAA T (SEQ ID NO: 1) and SK39 Primer: TTT GGT CCT TGT CTT ATG TCC AGA ATG
C (SEQ ID NO: 2)); and (iii) measuring the presence or absence of a
mutation at codon 68 of the HIV-RT, wherein the presence of the mutation
correlates positively with immunologic decline of the HIV-infected
individual over time. In other embodiments, the sample from the
HIV-infected individual is derived from or comprises lymphocytic cells
and step (ii) comprises amplifying HIV proviral DNA sequences without a
required conversion of RNA to cDNA. In preferred embodiments of the above
methods, the HIV primers used comprise SK38 Primer: ATA ATC CAC CTA TCC
CAG TAG GAG AAA T (SEQ ID NO: 1) and SK39 Primer: TTT GGT CCT TGT CTT ATG
TCC AGA ATG C (SEQ ID NO: 2), and/or the presence of HIV sequence is
detected using, e.g., an enzyme-linked assay (e.g., a colorimetric or
fluorescence based assay). The presence of the amplified product may also
be detected with the SK19 probe: ATC CTG GGA TTA AAT AAA ATA GTA AGA ATG
TAT AG (SEQ ID NO: 3). Similar methods in which the HIV copy number is
measured are also provided.

[0087] The presence of the codon 68, e.g., S68del, mutation indicates that
the effectiveness of monotherapy with DFC is likely to decline either in
the presence or the absence of the codon 68 mutation. Combination therapy
with DFC (e.g., by adding AZT) or a switch to other drugs as provided
herein is desirable.

Kits

[0088] The present invention also provides a kit for detection of
mutations at codon 68 (e.g., S68del) of HIV-RT encoding sequences.

[0089] In certain embodiments, the kit comprises a first pair of PCR
primers which bind outside the region of codon 68 and therefore may be
used to amplify a DNA fragment comprising codon 68 (e.g. SK38 Primer: ATA
ATC CAC CTA TCC CAG TAG GAG AAA T (SEQ ID NO: 1) and SK39 Primer: TTT GGT
CCT TGT CTT ATG TCC AGA ATG C (SEQ ID NO: 2)); and at least two pairs of
second round primers which may be used to amplify selectively codon 68,
e.g., S68del, sequences. The kit may include more than two pairs of
second primers. Similar primers may be readily designed by those skilled
in the art; the first pair of primers need only amplify a
conveniently-sized DNA fragment comprising codon 68 of HIV-RT, and one
member of the second pair of primers should bind selectively to codon 68,
preferably having its 3' terminus at the codon of interest in order to
maximize the probability of a perfect match resulting in amplification.
The kit may also include a probe for detection of the amplified product
containing codon 68, such as the SK19 probe: ATC CTG GGA TTA AAT AAA ATA
GTA AGA ATG TAT AG (SEQ ID NO: 3). Optionally, the kit may include
instructions for interpretation indicting that the presence of the mutant
form at the codon 68 of HIV-RT correlates with reduced efficacy of a
particular antiretroviral therapeutic agent, e.g., that presence of the
codon 68 mutant indicates reduced efficacy of monotherapy with DFC and a
number of other NRTI, including but not necessarily limited to
lamivudine, emtricitabine, tenofovir, abacavir and amdoxovir.

[0091] As detailed above, it is possible to study the quantity and/or
quality (such as screening for mutations) of HIV-specific DNA or RNA
sequences isolated from HIV-infected individuals (e.g., plasma samples or
lymphocytic cells such as PBM cells) to evaluate whether a particular
antiretroviral agent or therapy is an effective one. Well-known
extraction and purification procedures are available for the isolation of
DNA from a sample. Proviral DNA, for example, can be isolated from
patient samples, such as from lymphocytic cells (e.g., PBM cells), by
digestion of HIV-infected cells with proteinase K in the presence of EDTA
and a detergent such as SDS, followed by extraction with phenol.

[0092] HIV-specific RNA can be isolated from samples such as plasma
samples or lymphocytic cells, e.g., PBM cells, using the following
methodology. Suitable infected cells are incubated for a period of time.
The cells are recovered by centrifugation. The cells are resuspended in
an RNA extraction buffer followed by digestion using a proteinase
digestion buffer and digestion with proteinase K. Proteins are removed in
the presence of a phenol/chloroform mixture. RNA can then be recovered
following further centrifugation steps. (Maniatis, T., et al, Molecular
Coning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory
Press, (1989)).

[0093] Although it is possible to use non-amplified nucleic acid, due to
the relative scarcity of nucleic acid in an HIV-1 sample, it is
preferable to amplify it. Nucleic acid may be selectively amplified using
the general technique of polymerase chain reaction (PCR), which is an in
vitro method for producing large amounts of specific nucleic acid
fragment of defined length and sequence from small amounts of a template.

[0094] A standard PCR comprises standard reactants, using Mg2+
concentration, oligonucleotide primers and temperature cycling conditions
for amplification of the HIV gene of interest, such as the HIV-RT gene,
using sequence specific primers. The primers are chosen such that they
will amplify the entire RT gene or a selected sequence which incorporates
nucleotides corresponding to a region of the wild-type DNA sequence of
HIV-1 that includes the codon which is mutated. In a preferred embodiment
of the invention, primers 38K and 39K (SK38 Primer: ATA ATC CAC CTA TCC
CAG TAG GAG AAA T (SEQ ID NO: 1) and SK39 Primer: TTT GGT CCT TGT CTT ATG
TCC AGA ATG C (SEQ ID NO: 2)) are used to amplify the RT gene.

[0095] RNA cannot be amplified directly by PCR. Its corresponding cDNA
must first be synthesized. Synthesis of cDNA is normally carried out by
primed reverse transcription reactions using primers, such as for
example, using oligo-dT primers which hybridize to polyA tails found at
the 3'-end of many eukaryotic RNA transcripts (PolII). Advantageously,
primers are chosen such that they will simplify the nucleic acid sequence
for RT or a selected sequence which incorporates nucleotides
corresponding to the region of RNA corresponding to the wild-type DNA
sequence or to the region of the mutant DNA sequence corresponding to the
68th codon of the reverse transcriptase region. This could be achieved by
preparing an oligonucleotide primer which is complementary to a region of
the RNA strand which is upstream of the corresponding sequence of the
wild-type DNA sequence. cDNA prepared by this methodology (see Maniatis,
T., et al., supra.) can then be used in the same way as for the DNA
already discussed.

[0096] The next stage of the methodology is to hybridize to the nucleic
acid an oligonucleotide which is complementary to a region of the
wild-type DNA sequence (or its corresponding RNA) or to a region of the
mutant DNA sequence (or its corresponding RNA).

[0097] Conditions and reagents are then provided to permit polymerization
of the nucleic acid from the 3'-end of the oligonucleotide primer. Such
polymerization reactions are well-known in the art.

[0098] If the oligonucleotide primer has at its 3'-end a nucleotide which
is complementary to a mutant genotype, that is a genotype which has a
nucleotide change at the 68th codon in the RT region, then polymerization
of the nucleic acid sequence will only occur if the nucleic acid of the
sample is the same as the mutant genotype. Polymerization of a wild type
nucleic acid sequence will not occur or at least not to a significant
extent because of a mis-match of nucleotides at the 3'-end of the
oligonucleotide primer and the nucleic acid sequence of the sample.

[0099] If the oligonucleotide primer has at its 3'-end of nucleotide which
is complementary to the wild-type genotype, that is a genotype which has
the wild-type nucleotide at the 68th codon in the RT region, then there
will be polymerization of a nucleic acid sequence which is wild-type at
that position. There will be no polymerization of a nucleic acid which
has a mutant nucleotide at the 3'-position.

[0101] It is convenient to determine the presence of an oligonucleotide
primer extended product. The means for carrying out the detection is by
using an appropriate label.

[0102] The label may be conveniently attached to the oligonucleotide
primer or to some other molecule which will bind the primer extended
polymerization product.

[0103] The label may be for example an enzyme, radioisotope or
fluorochrome. A preferred label may be biotin which could be subsequently
detected using streptavidin conjugated to an enzyme such as peroxidase or
alkaline phosphatase. The presence of an oligonucleotide primer extended
polymerization product can be detected for example by running the
polymerization reaction on an agarose gel and observing a specific DNA
fragment labeled with ethidium bromide, or Southern blotted and
autoradiographed to detect the presence or absence of bands corresponding
to polymerized product. If a predominant band is present which
corresponds only to the labeled oligonucleotide then this indicates that
polymerization has been occurred. If bands are present of the correct
predicted size, this would indicate that polymerization has occurred.

[0104] For example, DNA isolated from HIV-infected individuals' plasma
samples or PBM cells as described herein is used as a template for PCR
amplification using synthetic oligonucleotide primers which either match
or mis-match with the amplified sequences. The feasibility of PCR in
detecting such mutations has already been demonstrated. PCR using the
Amplification Refractory Mutation system ("ARMS") (Newton, C. R., et al.
Nucleic Acids Research, 17, p 2503, (1989)) Synthetic oligonucleotide are
produced that anneal to the regions adjacent to an including the specific
mutations such that the 3'-end of the oligonucleotide either matches of
mismatches with a mutant or wild-type sequence. PCR is carried out which
results in the identification of a DNA fragment (using gel
electrophoresis) where a match has occurred or no fragment where a
mismatch occurred.

[0105] DNA is extracted from HIV-1 infected T-cells as described herein
and subjected to "ARMS" PCR analysis using these primers.

[0106] The presence of a fragment is identified by using an
oligonucleotide primer as described above, i.e., by attempting
polymerization using an oligonucleotide primer which may be labeled for
the amplified DNA fragment under stringent conditions which only allow
hybridization of complementary DNA (the only difference is that
differential hybridization does not have to be performed as fragments of
DNA amplified by the "ARMS" method will be the same whether derived from
mutant or wild-type DNS, so a common oligonucleotide can be used to
detect the presence of these fragments. The sequence of such an
oligonucleotide is derived from a DNA sequence within the DNA fragment
that is conserved amongst HIV-1 strains).

[0107] The above PCR assay may be adapted to enable direct detection of
mutations associated with D-D4FC resistance in DNA from PBL samples from
infected individuals that have not been cultured to obtain virus. As this
material generally contains considerably less HIV-1 DNA than that in
infected lymphoid cultures a "double PCR" (or nested set) protocol can be
used (Simmonds et al., J. Virol., 64, 864-872, (1990)) to boost the
amount of target HIV-1 RT DNA signal in the samples. The double PCR
overcomes the problem of limited amplification of a rare template
sequence. A small amount of the pre-amplified material may be used in the
second PCR with primer pairs designed to allow discrimination of wild
type and mutant residues.

[0108] The presence of a codon 68 mutation in RT can also be determined by
quantitative real-time PCR, as described in Example 4.

[0109] It is also possible to detect codon 68 mutations in the HIV-1 RT
RNA isolated from clinical samples using an RNA amplification system.
Using the methodology described by Guatelli et al. (Proc. Natl. Acad.
Sci, (USA), 8/7, 1874-1878, (March 1990)) a target nucleic acid sequence
can be replicated (amplified) exponentially in vitro under isothermal
conditions by using three enzymatic activities essential to retroviral
replication: reverse transcriptase, RNase H and a DNA-dependant RNA
polymerase. Such a methodology may be employed followed by an
hybridization step to distinguish mutant from wild-type nucleotides at
discussed previously.

[0110] The viral RNA or corresponding DNA from an HIV-infected person may
be directly assayed. Alternatively, part or all of the HIV-RT encoding
sequences may be cloned into viral vectors and amplified to produce
larger amounts of viral nucleic acid for sequencing and other desired
analyses.

[0113] In certain embodiments of the invention, the presence of the S68del
mutation may be detected by solid-state nucleic acid sensors. In specific
embodiments, the solid-state sensors are oligonucleotide microarrays,
cDNA microarrays and nucleic acids bound to any other convenient solid
supports, such as beads or other microspheres. Examples of such sensors
are further described in Sievertzon et al., Expert Rev. Mol. Diagn. 2006;
6:481-492; Heller, Annu. Rev. Biomed. Eng. 2002; 4:129-153; and Watson et
al., Curr. Opinion in Biotech. 1998; 9:609-614.

[0114] In one specific embodiment, a line probe assay can be used to
detect the codon 68, e.g., S68del, mutation in samples collected from
HIV-infected individuals. Oligonucleotide probes used to detect the
S68del mutation, for example, are applied to a nitrocellulose or other
suitable membrane. RNA or DNA isolated from HIV-infected individuals is
amplified and labeled, for example, by biotinylation. The labeled nucleic
acid is reverse hybridized to the probes, and the amount of hybridized
nucleic acid is detected. Details of probe itemization, nitrocellulose
strip production and reverse hybridization have been published previously
(Stuyver et al., J. Clin. Microbiol. 1996; 34:2259-2266; Stuyver et al.,
Antimicrob. Agents Chemother. 1997; 41:284-291; Van Geyt et al., in
Therapies of Viral Hepatitis 1998; 139-145).

[0115] Any of a number of available systems and assays may be used in
conjunction with products and methods of the invention to assess viral
genotypes and associated phenotypes such as antiretroviral drug
susceptibility, including but not limited to certain commercially
available systems (see, e.g., PhotoSense® HIV (Monogram Biosciences);
HIV GenoSure® (LabCorp; see also Baxter et al., AIDS 2000 14(9):F83-93
(2000) and Durant et al., Lancet 353(9171):2195-2199 (1999);
Antivirogram® (Vireo) and Kellam, Antimicrob. Agents Chemother.
38:23-30 (1994)).

[0116] In other embodiments, the S68del mutation sequence and drug
resistance profile may be added to HIV genotyping and phenotyping
databases. Samples isolated from an HIV-infected individual may then be
compared to such databases to correlate the viral genotype and/or viral
phenotype of the individual sample to effective antiretroviral therapies.
In particular, such methods comprising comparison to information stored
in a database may be used to choose effective NRTI treatment (including
NRTI in combination with one or more other NRTI and/or other agents)
(see, e.g., Lengauer et al., Nature Rev. Microbiol. 4:790-797 (2006);
Baxter et al., AIDS 2000 14:F83-F93 (2000); and Durant et al., Lancet
353(9171):2195-2199 (1999)).

[0117] Alternatively, the HIV RT protein may be screened directly or
indirectly for the mutation using any of a number of available protein
sequence based techniques. Such techniques include protein expression
based assays, optionally in combination with Western blotting techniques.
In certain embodiments, a denatured form of the HIV RT protein containing
the S68del mutation may be used to raise antibodies that bind
differentially to denatured S68del and the wild-type RT proteins (or
fragments thereof comprising the S68 codon). A variety of protein
expression systems are known and available to the skilled worker. The RT
may be expressed in a baculovirus system, for example. Antibodies having
specificity for an S68del specific epitope that may be engineered include
monoclonal, chimeric, humanized or human antibodies, and also include any
number of antibody fragments and single chain antibodies. The antibody
that binds the S68del and the wild-type forms of RT differentially can be
used in Western blots, ELISAs and other immunoassays to detect the
presence of the S68del HIV mutation in samples from HIV-infected
individuals.

Methods to Avoid Selecting, or to Treat an Individual Harboring, HIV with
a Codon 68 Mutation in HIV-RT

[0118] The invention further provides methods for treating a subject
infected with HIV-1 or HIV-2 comprising the step of administering over
time an antiretroviral agent that does not select for an HIV-1 mutant
having a codon 68 mutation in the HIV reverse transcriptase coding
sequence.

[0120] In another embodiment, the antiretroviral agent administered to
avoid selecting HIV-1 with a codon 68 mutation is an NNRTI. Examples of
NNRTI include, but are not limited to, DMP-266
((S)-6-chloro-4-cyclopropylethynyl-4-trifluoromethyl-1,4-dihydro-2H-3,1-b-
-enzoxazin-2-one (SUSTIVA, see U.S. Pat. No. 5,519,021); delavirdine,
(1-[3-(1-methyl-ethyl)amino]-2-pyridinyl-4-[[5-[(methylsulfonyl)amino]-1H-
-indol-2-yl]carbonyl]-, monoethanesulfonate), nevirapine, or delarvirdine.

[0121] In other embodiments, HIV-infected individuals can be treated with
NRTI against which the S68del mutant does not show increased resistance.
Examples of such NRTI include AZT, DDI, DFDOC, D4T, DOT and DDC.

[0122] In other embodiments, the antiretroviral agent administered to
avoid selecting HIV-1 with a codon 68 mutation is an HIV fusion
inhibitor, an HIV integrase inhibitor, an RNAse H inhibitor, a CD4
binding inhibitor, a CXCR4 binding inhibitor, or a CCR5 binding
inhibitor.

[0123] The present invention further provides methods for isolating
compounds that are active against an HIV-1 S68del mutant using the
screening methods and the S68del mutant HIV of the invention. A variety
of protocols for characterizing antiretroviral agents and their effects
on viral replication, in vitro and in vivo, such as those described and
exemplified herein, are well known in the literature. See, e.g.,
Lennerstrand et al., Antimicrob. Agents Chemother. 2007 Apr. 2 (Epub
ahead of print); Hammond et al., Antimicrob. Agents Chemother.
49(9):3930-3932 (2005); Moser et al., Antimicrob. Agents Chemother.
49(8):3334-3340 (2005); Parikh et al., Antimicrob. Agents Chemother.
49(3):1139-1144 (2005); Boyer et al., J. Virol. 78(18):9987-9997 (2004);
Roge et al., Antiviral Therapy 8:173-182 (2003); Boyer et al., J. Virol.
76(18):9143-9151 (2002); Van Vaerenbergh, Verh. K Acad. Geneeskd. Belg.
63(5):447-473 (2001); Tamalet et al., Virol. 270:310-316 (2000); and
Bazmi et al., Antimicrob. Agents Chemother. 44(7):1783-1788 (2000); each
incorporated herein by reference. These or similar methods may be used to
characterize known antiretroviral compounds and to identify and isolate
new antiretroviral compounds that are useful in the treatment of
multidrug resistant HIV-1, such as the HIV-1 S68del mutant of the
invention.

[0124] The dosages for such antiretroviral agents will depend on such
factors as absorption, biodistribution, metabolism and excretion rates
for each drug as well as other factors known to those of skill in the
art. It is to be noted that dosage values will also vary with the
severity of the condition to be alleviated. It is to be further
understood that for any particular subject, specific dosage regimens and
schedules should be adjusted over time according to the individual need
and the professional judgment of the person administering or supervising
the administration of the compositions. Examples of suitable dosage
ranges for anti-HIV compounds, including nucleoside derivatives or
protease inhibitors can be found in the scientific literature and in the
Physicians Desk Reference. Many examples of suitable dosage ranges for
other compounds described herein are also found in public literature or
can be identified using known procedures. These dosage ranges can be
modified as desired to achieve a desired result.

Preparation of Pharmaceutical Compositions

[0125] Any antiretroviral agent described herein can be administered to
the HIV-infected individual as a pharmaceutically acceptable salt or
prodrug in the presence of a pharmaceutically acceptable carrier or
diluent, for any of the indications or modes of administration as
described in detail herein. The active materials can be administered by
any appropriate route, for example, orally, parenterally, enterally,
intravenously, intradermally, subcutaneously, transdermally, intranasally
or topically, in liquid or solid form.

[0126] The active compound(s) are included in the pharmaceutically
acceptable carrier or diluent in an amount sufficient to deliver to a
HIV-infected individual a therapeutically effective amount of compound to
inhibit viral replication in vivo, especially HIV replication, without
causing serious toxic effects in the treated individual. By "inhibitory
amount" is meant an amount of active ingredient sufficient to exert an
inhibitory effect on viral replication as measured by, for example, an
assay such as the ones described herein. Preferably, inhibitory effect is
at least 2.5-fold, and preferably at least 4-fold, 5-fold, 7-fold,
10-fold or higher.

[0127] A preferred dose of the compound for all the above-mentioned
conditions will be in the range from about 1 to 75 mg/kg, preferably 1 to
20 mg/kg, of body weight per day, more generally 0.1 to about 100 mg per
kilogram body weight of the recipient per day. The effective dosage range
of the pharmaceutically acceptable derivatives can be calculated based on
the weight of the parent nucleoside to be delivered. If the derivative
exhibits activity in itself, the effective dosage can be estimated as
above using the weight of the derivative, or by other means known to
those skilled in the art.

[0128] The compounds are conveniently administered in unit any suitable
dosage form, including but not limited to one containing 7 to 3000 mg,
preferably 70 to 1400 mg of active ingredient per unit dosage form. An
oral dosage of 50 to 1000 mg is usually convenient.

[0129] Ideally, the active ingredient should be administered to achieve
peak plasma concentrations of the active compound of from about 0.02 to
70 micromolar, preferably about 0.5 to 10 mM. This may be achieved, for
example, by the intravenous injection of a 0.1 to 25% solution of the
active ingredient, optionally in saline, or administered as a bolus of
the active ingredient.

[0130] The concentration of active compound in the drug composition will
depend on absorption, distribution, metabolism and excretion rates of the
drug as well as other factors known to those of skill in the art. It is
to be noted that dosage values will also vary with the severity of the
condition to be alleviated. It is to be further understood that for any
particular subject, specific dosage regimens should be adjusted over time
according to the individual need and the professional judgment of the
person administering or supervising the administration of the
compositions, and that the concentration ranges set forth herein are
exemplary only and are not intended to limit the scope or practice of the
claimed composition. The active ingredient may be administered at once,
or may be divided into a number of smaller doses to be administered at
varying intervals of time.

[0131] A preferred mode of administration of the active compound is oral.
Oral compositions will generally include an inert diluent or an edible
carrier. They may be enclosed in gelatin capsules or compressed into
tablets. For the purpose of oral therapeutic administration, the active
compound can be incorporated with excipients and used in the form of
tablets, troches, or capsules. Pharmaceutically compatible bind agents,
and/or adjuvant materials can be included as part of the composition.

[0132] The tablets, pills, capsules, troches and the like can contain any
of the following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as magnesium
stearate or Sterotes; a glidant such as colloidal silicon dioxide; a
sweetening agent such as sucrose or saccharin; or a flavoring agent such
as peppermint, methyl salicylate, or orange flavoring. When the dosage
unit form is a capsule, it can contain, in addition to material of the
above type, a liquid carrier such as a fatty oil. In addition, dosage
unit forms can contain various other materials which modify the physical
form of the dosage unit, for example, coatings of sugar, shellac, or
other enteric agents.

[0133] The compounds can be administered as a component of an elixir,
suspension, syrup, wafer, chewing gum or the like. A syrup may contain,
in addition to the active compounds, sucrose as a sweetening agent and
certain preservatives, dyes and colorings and flavors.

[0134] The compounds or their pharmaceutically acceptable derivative or
salts thereof can also be mixed with other active materials that do not
impair the desired action, or with materials that supplement the desired
action, such as antibiotics, antifungals, antiinflammatories, protease
inhibitors, or other nucleoside or non-nucleoside antiviral agents, as
discussed in more detail above. Solutions or suspensions used for
parental, intradermal, subcutaneous, or topical application can include
the following components: a sterile diluent such as water for injection,
saline solution, fixed oils, polyethylene glycols, glycerine, propylene
glycol or other synthetic solvents; antibacterial agents such as benzyl
alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfate; chelating agents such as ethylenediaminetetraacetic acid;
buffers such as acetates, citrates or phosphates and agents for the
adjustment of tonicity such as sodium chloride or dextrose. The parental
preparation can be enclosed in ampoules, disposable syringes or multiple
dose vials made of glass or plastic.

[0136] Liposomal suspensions (including liposomes targeted to infected
cells with monoclonal antibodies to viral antigens) are also preferred as
pharmaceutically acceptable carriers these may be prepared according to
methods known to those skilled in the art, for example, as described in
U.S. Pat. No. 4,522,811 (which is incorporated herein by reference in its
entirety). For example, liposome formulations may be prepared by
dissolving appropriate lipid(s) (such as stearoyl phosphatidyl
ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl
choline, and cholesterol) in an inorganic solvent that is then
evaporated, leaving behind a thin film of dried lipid on the surface of
the container. An aqueous solution of the active compound or its
monophosphate, diphosphate, and/or triphosphate derivatives is then
introduced into the container. The container is then swirled by hand to
free lipid material from the sides of the container and to disperse lipid
aggregates, thereby forming the liposomal suspension.

Controlled Release Formulations

[0137] Any antiretroviral agent described herein can be administered as a
controlled release formulation. The field of biodegradable polymers has
developed rapidly since the synthesis and biodegradability of polylactic
acid was reported by Kulkarni et al., in 1966 (Arch. Surg., 93:839).
Examples of other polymers which have been reported as useful as a matrix
material for delivery devices include polyanhydrides, polyesters such as
polyglycolides and polylactide-co-glycolides, polyamino acids such as
polylysine, polymers and copolymers of polyethylene oxide, acrylic
terminated polyethylene oxide, polyamides, polyurethanes,
polyorthoesters, polyacrylonitriles, and polyphosphazenes. See, for
example, U.S. Pat. Nos. 4,891,225 and 4,906,474 (polyanhydrides), U.S.
Pat. No. 4,767,628 (polylactide, polylactide-co-glycolide acid), and U.S.
Pat. No. 4,530,840, et al. (polylactide, polyglycolide, and copolymers).
See also U.S. Pat. No. 5,626,863 which describes photopolymerizable
biodegradable hydrogels as tissue contacting materials and controlled
release carriers (hydrogels of polymerized and crosslinked macromers
comprising hydrophilic oligomers having biodegradable monomeric or
oligomeric extensions, which are end capped monomers or oligomers capable
of polymerization and crosslinking); and PCT WO 97/05185 directed to
multiblock biodegradable hydrogels for use as controlled release agents
for drug delivery and tissue treatment agents.

[0138] Degradable materials of biological origin are well known, for
example, crosslinked gelatin. Hyaluronic acid has been crosslinked and
used as a degradable swelling polymer for biomedical applications (U.S.
Pat. No. 4,957,744).

[0139] Many dispersion systems are currently in use as, or being explored
for use as, carriers of substances, particularly biologically active
compounds. Dispersion systems used for pharmaceutical and cosmetic
formulations can be categorized as either suspensions or emulsions.
Suspensions are defined as solid particles ranging in size from a few
manometers up to hundreds of microns, dispersed in a liquid medium using
suspending agents. Solid particles include microspheres, microcapsules,
and nanospheres. Emulsions are defined as dispersions of one liquid in
another, stabilized by an interfacial film of emulsifiers such as
surfactants and lipids. Emulsion formulations include water in oil and
oil in water emulsions, multiple emulsions, microemulsions,
microdroplets, and liposomes. Microdroplets are unilamellar phospholipid
vesicles that consist of a spherical lipid layer with an oil phase
inside, as defined in U.S. Pat. Nos. 4,622,219 and 4,725,442. Liposomes
are phospholipid vesicles prepared by mixing water-insoluble polar lipids
with an aqueous solution. The unfavorable entropy caused by mixing the
insoluble lipid in the water produces a highly ordered assembly of
concentric closed membranes of phospholipid with entrapped aqueous
solution.

[0140] U.S. Pat. No. 4,938,763 discloses a method for forming an implant
in situ by dissolving a nonreactive, water insoluble thermoplastic
polymer in a biocompatible, water soluble solvent to form a liquid,
placing the liquid within the body, and allowing the solvent to dissipate
to produce a solid implant. The polymer solution can be placed in the
body via syringe. The implant can assume the shape of its surrounding
cavity. In an alternative embodiment, the implant is formed from
reactive, liquid oligomeric polymers which contain no solvent and which
cure in place to form solids, usually with the addition of a curing
catalyst.

[0141] A number of patents disclose drug delivery systems that can be used
to administer D-D4FC or a nucleotide or other defined prodrug thereof
U.S. Pat. No. 5,749,847 discloses a method for the delivery of
nucleotides into organisms by electrophoration. U.S. Pat. No. 5,718,921
discloses microspheres comprising polymer and drug dispersed there
within. U.S. Pat. No. 5,629,009 discloses a delivery system for the
controlled release of bioactive factors. U.S. Pat. No. 5,578,325
discloses nanoparticles and microparticles of non-linear hydrophilic
hydrophobic multiblock copolymers. U.S. Pat. No. 5,545,409 discloses a
delivery system for the controlled release of bioactive factors. U.S.
Pat. No. 5,494,682 discloses ionically cross-linked polymeric
microcapsules.

[0142] U.S. Pat. No. 5,728,402 describes a controlled release formulation
that includes an internal phase which comprises the active drug, its salt
or prodrug, in admixture with a hydrogel forming agent, and an external
phase which comprises a coating which resists dissolution in the stomach.
U.S. Pat. Nos. 5,736,159 and 5,558,879 discloses a controlled release
formulation for drugs with little water solubility in which a passageway
is formed in situ. U.S. Pat. No. 5,567,441 discloses a once-a-day
controlled release formulation. U.S. Pat. No. 5,508,040 discloses a
multiparticulate pulsatile drug delivery system. U.S. Pat. No. 5,472,708
discloses a pulsatile particle based drug delivery system. U.S. Pat. No.
5,458,888 describes a controlled release tablet formulation which can be
made using a blend having an internal drug containing phase and an
external phase which comprises a polyethylene glycol polymer which has a
weight average molecular weight of from 3,000 to 10,000. U.S. Pat. No.
5,419,917 discloses methods for the modification of the rate of release
of a drug form a hydrogel which is based on the use of an effective
amount of a pharmaceutically acceptable ionizable compound that is
capable of providing a substantially zero-order release rate of drug from
the hydrogel. U.S. Pat. No. 5,458,888 discloses a controlled release
tablet formulation.

[0143] U.S. Pat. No. 5,641,745 discloses a controlled release
pharmaceutical formulation which comprises the active drug in a
biodegradable polymer to form microspheres or nanospheres. The
biodegradable polymer is suitably poly-D,L-lactide or a blend of
poly-D,L-lactide and poly-D,L-lactide-co-glycolide. U.S. Pat. No.
5,616,345 describes a controlled absorption formulation for once a day
administration that includes the active compound in association with an
organic acid, and a multi-layer membrane surrounding the core and
containing a major proportion of a pharmaceutically acceptable
film-forming, water insoluble synthetic polymer and a minor proportion of
a pharmaceutically acceptable film-forming water soluble synthetic
polymer. U.S. Pat. No. 5,641,515 discloses a controlled release
formulation based on biodegradable nanoparticles. U.S. Pat. No. 5,637,320
discloses a controlled absorption formulation for once a day
administration. U.S. Pat. Nos. 5,580,580 and 5,540,938 are directed to
formulations and their use in the treatment of neurological diseases.
U.S. Pat. No. 5,533,995 is directed to a passive transdermal device with
controlled drug delivery. U.S. Pat. No. 5,505,962 describes a controlled
release pharmaceutical formulation.

Prodrug Formulations

[0144] Any of antiretroviral agents which are described herein can be
administered as an acylated prodrug or a nucleotide prodrug, as described
in detail below.

[0145] Any of the nucleosides described herein or other compounds that
contain a hydroxyl or amine function can be administered as a nucleotide
prodrug to increase the activity, bioavailability, stability or otherwise
alter the properties of the nucleoside. A number of nucleotide prodrug
ligands are known. In general, alkylation, acylation or other lipophilic
modification of the hydroxyl group of the compound or of the mono, di or
triphosphate of the nucleoside will increase the stability of the
nucleotide. Examples of substituent groups that can replace one or more
hydrogens on the phosphate moiety or hydroxyl are alkyl, aryl, steroids,
carbohydrates, including sugars, 1,2-diacylglycerol and alcohols. Many
are described in R. Jones and N. Bischofberger, Antiviral Research, 27
(1995) 1-17. Any of these can be used in combination with the disclosed
nucleosides or other compounds to achieve a desired effect.

[0147] Non-limiting examples of U.S. patents that disclose suitable
lipophilic substituents that can be covalently incorporated into the
nucleoside or other hydroxyl or amine containing compound, preferably at
the 5'-OH position of the nucleoside or lipophilic preparations, include
U.S. Pat. Nos. 5,149,794; 5,194,654 5,223,263; 5,256,641; 5,411,947;
5,463,092; 5,543,389; 5,543,390; 5,543,391; and 5,554,728, each of which
is incorporated herein by reference. Foreign patent applications that
disclose lipophilic substituents that can be attached to the nucleosides
of the present invention, or lipophilic preparations, include WO
89/02733, WO 90/00555, WO 91/16920, WO 91/18914, WO 93/00910, WO
94/26273, WO 96/15132, EP 0 350 287, EP 93917054.4, and WO 91/19721.

[0151] All publications and patents cited are hereby incorporated by
reference in their entirety.

[0152] Throughout this specification and paragraphs, the word "comprise"
or variations such as "comprises" or "comprising" will be understood to
imply the inclusion of a stated integer or group of integers but not the
exclusion of any other integer or group of integers.

[0153] The following are examples which illustrate the compositions and
methods of this invention. These examples should not be construed as
limiting: the examples are included for the purposes of illustration
only. This invention has been described with reference to its preferred
embodiments. Variations and modifications of the invention, will be
obvious to those skilled in the art from the foregoing detailed
description of the invention. It is intended that all of these variations
and modifications be included within the scope of this invention.

[0159] HIV-1/LAI obtained from the Centers for Disease Control and
Prevention (Atlanta, Ga.) was used as the virus for the resistant pool. A
multiplicity of infection (MOI) of 0.1, as determined by a limiting
dilution method in PBM cells, was selected to begin the infected pool.

[0160] Selection of Resistant Virus.

[0161] Naive PBM cells were treated with DFC at 0.1 μM for one hour
prior to inoculation with HIV-1/LAI. The treated PBM cell group and a
control nontreated PBM cell group were allowed to infect for 1 hr. IL-2
(26 IU/ml)-supplemented RPMI-1640 was then added for a final
concentration of 1×106 cells/ml. Virus was passaged every 6
days with a fresh treatment of DFC, ranging from 0.1 μM to 6 μM
over 52 weeks. RT activity was measured weekly and used to determine
percent inhibition of DFC. Total RNA was isolated from culture
supernatants using the commercial QIAmp Viral RNA mini kit (Qiagen,
Valencia, Calif.). Reverse transcriptase PCR was performed using
Invitrogen Superscript Reverse Trancriptase III to generate second strand
cDNA from viral RNA using Ambion DECAprime II primers. PCR was performed
using Invitrogen Platinum Taq polymerase (high fidelity). A 1346 bp
fragment of the HIV-1 genome was amplified using forward primer
5'-ttgactcagattggttgcactttaa-3' (SEQ ID NO: 4) and reverse primer
5'-aagaacccatagtaggagcagaaac-3' (SEQ ID NO: 5). The PCR product was
purified using the QIAquick PCR purification kit. The samples were
sequenced in both directions for the HIV-1 RT amino acids 01-300.
Sequencing was performed in parallel between the control virus and DFC
treated virus to determine if there were any mutations created by the
applied drug pressure on weeks when the virus appeared to be resistant
(Table 1 (WT--wild type); FIG. 1).

[0162] Population sequencing of virus during this assay revealed a
disruption of the S68 codon in the reverse transcriptase (RT) sequence,
which may alternatively be a deletion of the AGT codon 68 trinucleotide,
or of the adjacent +1 frameshift trinucleotide GTA (Table 2).

[0163] The S68del mutation was first detected by population sequencing at
week 14 as a mix with wild-type (WT) (Table 1). By week 19, S68del
dominated the pool. At week 25, some K65R was detected as well. In week
28, only K65R was detectible. At week 52, the pool contained a mixture of
S68del and K65R.

[0164] Cloning of the S68del virus demonstrated that S68del can occur
independently or as a mixture with wild-type, K65R, T69A or T69S.
Sometimes the mutations can be found in the same genome. No other
mutation in the reverse transcriptase region was detected.

[0166] Mutations that occur in the HIV-1 RT region between amino acids 62
and 78 increase NRTI resistance significantly (Hu et al., J. Acquir.
Immune Defic. Syndr. 2007; 45:494-500). Drug resistance of the S68del
virus isolated at week 23 (HIV.sub.s68Δ-23) was measured with the
3H-TTP RT incorporation assay in human PBM cells (Schinazi, et al.,
Antimicrob. Agents Chemother. 1990; 34:1061-1067; Stuyver et al.,
Antimicrob. Agents Chemother. 2002; 46:3854-60). HIV.sub.s68Δ-23
was population sequenced to ensure the dominant population was the
deletion at codon 68. TOPO® cloning (Invitrogen) performed on
HIV.sub.s68Δ-23 indicated that approximately 90% of the population
was pure S68del. The other approximately 10% either had a T69 deletion or
S68del with a mutation T69A. The susceptibility of the S68del virus to
several nucleoside reverse transcriptase inhibitors (NRTI), a
non-nucleoside reverse transcriptase inhibitor (NNRTI) and a protease
inhibitor (PI) was tested. Fold increases were measured relative to
HIVLAI (Table 4, FIGS. 2 and 3). Data are the averages of 2-6
independent experiments.

[0169] A site directed S68 deletion mutant was created by digesting pCR
2.1 (Invitrogen) and pNL4-3 (AF3244930) with restriction enzymes Eco RI
and Spe I. The pCR2.1 was cut into a single band of 3.9 kb, and the
pNL4-3 was cut into one 4.2 kb band and one 10.6 kb band. Nucleic acids
were separated by gel electrophoresis in a TAE gel and extracted from
bands using the Qiagen gel extraction kit. The 4.2 kb band from pNL4-3
containing the HIV-RT encoding sequences was ligated into the linearized
pCR2.1, making the construct MC002. Site directed mutagenesis was
performed on the MC002 plasmid. Primers used had the S68 codon AGT
deleted, thus introducing the S68 deletion into the pNL4-3 RT background.
Presence of the S68 codon AGT deletion (and no other surrounding
mutations) was confirmed by sequencing in both directions.

[0170] To ligate the RT of pNL4-3 comprising the deletion at S68 codon 68
into protein expression vector pE60 (Invitrogen), MC002 was amplified
using forward primer 5'-CGCGCCCATGGTGCCCATTAGTCCTATTGAGACTGTACC-3' (SEQ
ID NO: 10) and reverse primer 5'-GCGCGCAGATCTTAGTACTTTCCTGATTCC
AGCACTGAC-3' (SEQ ID NO: 11). The PCR product and pQE60 were digested
with Bgl II and Nco I. The digested products were separated by gel
electrophoresis nucleic acids in excised gel bands extracted using the
Qiagen gel extraction kit. The amplified PCR product comprising the RT of
pNL4-3 was ligated into the linearized pQE60. The ligation mix was
transformed into chemically competent bacteria (Alpha-select). Plasmid
DNA from selected transformants was extracted using a mini-prep kit
(Qiagen). Plasmid DNA was sequenced in both directions to confirm the
ligation.

[0172] S68delpNL4-3 showed increased resistance against DFC, but not
against AZT. These results confirmed results obtained by the 3H-TTP
RT incorporation assay and the heteropolymeric-DNA colorimetric RT assay
with the in vitro selected S68del virus. Thus, the results show that the
S68delpNL4-3 virus will be a useful and valid construct to measure
S68del drug resistance phenotypes. Furthermore, the real-time PCR assay
will serve as an accurate method for measuring drug resistance. Finally,
these results confirm that the deletion of the AGT codon 68
trinucleotide, or deletion of the adjacent +1 frameshift trinucleotide
GTA, is solely responsible for increased resistance of the virus to
growth in the presence of DFC.

[0173] The principle and performance of the non-radioactive RT assay has
been described (Lennerstrand et al., Antimicrob. Agents Chemother. 2007;
51:2078-2084). Separate kit components, such as covalently-linked DNA
microtiter plates and tracer solution (alkaline phosphate (AP)-conjugated
anti-BrdU antibody) were obtained from Cavidi Tech, Uppsala, Sweden. In
brief, the 96-well microtiter plate used consisted of an 18 base
heteropolymeric DNA primer covalently bound to the well. In the RT assay,
the DNA primer is bound to a 50-base DNA template at 50 ng/well (190 nM)
with a 5'-A12-3' tail with a 5'-(GTCA)5-3' repeat (Integrated
DNA Technologies, USA). The RT assay reaction mixture (total volume 150
μl/well) contained: Hepes 50 mM, pH 7.3; MgCl2 10 mM; Triton
X-100 0.5%; bovine serum albumin 0.1 mg/ml; dATP, dGTP, dCTP and
5-bromo-2'-deoxyuridine-5'-triphosphate (BrdUTP) at 1.0 μM each (where
BrdUTP replaces TTP) (Sigma). To obtain ATP primer unblocking reaction in
the assay, the ATP (Amersham/GE Health Care) was set to physiological
concentration (3.2 mM). However, the ATP was only used in the assay with
virus pellets as sample, not merely for primer unblocking reaction but to
protect degradation of substrate in the crude sample. Furthermore, the
dNTP level including BrdUTP was increased from 1 μM to 4 μM for the
assay with the virus pellet samples. Subsequently, in the assay with
recombinant purified RT enzyme, no ATP besides 1 μM dNTP was used.

[0174] The reaction was started with the addition of RT either as
recombinant or crude virus pellet form in a similar activity range as
previous published (Lennerstrand et al., Antimicrob. Agents Chemother.
2007; 51:2078-2084). The RT reaction mixture was incubated at 33°
C. for 180 min and terminated by NaOH (to dehybridize the template) and
water washing of the plates. The tracer incubation step with anti-BrdU
antibodies-AP-conjugated and the detection step for color absorbance at
405 nm was performed as previously described (Lennerstrand et al.,
Antimicrob. Agents Chemother. 2007; 51:2078-2084. The NRTI-TP used were
AZT-TP (Cavidi Tech), DFC-TP and (-) FTC-TP. The latter nucleotides were
synthesized from the corresponding nucleoside analog (Ludwig et al., J.
Org. Chem. 1989; 54:631-635).

[0175] The resistance of RT derived from S68del and M184V particles
against AZT, (-)FTC and DFC triphosphates was tested by a
heteropolymeric-DNA colorimetric RT assay with 3.2 mM ATP. Fold-increases
were measured relative to HIVLAI (FIG. 5). Both S68del and M184V RTs
showed increased resistance to (-)FTC. Only S68del RT showed increased
resistance to DFC (FIG. 5). RT from virally-derived S68del demonstrated a
5.6-, 2.5- and 10-fold increase in resistance to DFC-TP, AZT-TP and
(-)FTC-TP, respectively, in the enzymatic assay (FIG. 6).

[0176] The level of resistance to NRTI-TP by the S68del mutants compared
to wild type RT was determined as IC50 values of RT activity in the
absence of ATP (Table 6). Fold-increased resistance values were
determined by dividing the IC50 for the mutant by the IC50 for
respective wild type. The RT activity was linear during the assay time
within the substrate range used, and thus steady state kinetics were
assumed.

[0177] Enzymatic studies of the S68del RT detected similar resistance to
NRTI-TP with and without ATP. Without being bound by theory, this result
suggests that S68del resistance is not ATP-dependent and most likely
occurs by enhanced substrate discrimination.